We have previously identified and characterized by cDNA cloning a third functional gene, designated erbB-3, in the human erbB/epidermal growth factor (EGF) subfamily of tyrosine kinases. Its predicted coding structure revealed features characteristic of a growth factor receptor tyrosine kinase with significantly greater overall similarities to both epidermal growth factor receptor (EGFR) and products than to any other tyrosine kinase. For high level in vitro expression of the encoded erbB-3 gene product. its complete coding sequence was introduced in LTR-based eukaryotic expression vectors and transfected in the presence of a selectable marker gene into NIH/3T3 fibroblasts. Utilizing polyclonal antisera raised against epitope-specific peptides in the predicted erbB-3 coding sequence, the mature erbB-3 product was identified as a 180-kDa protein in marker-selected transfectants containing the recombinant expression vector and was absent from control NIH/3T3 cells. Biochemical characterization of the erbB-3 protein showed its biosynthesis as a 145-kDa precursor polypeptide which becomes posttranslationally modified by N-linked glycosylation into a 180-kDa mature glycoprotein. Immunohistochemical analysis with epitope-specific antisera identified a transmembrane topology of the mature erbB-3 protein. Its ability to autophosphorylate on tyrosine residues demonstrated an intrinsic tyrosine kinase activity associated with the cytoplasmic portion of the gpl80erbB-3 protein. Preliminary binding and triggering studies did not reveal direct interaction of EGF or transforming growth factor alpha (TGFalpha). the two prototype ligands for the closely related EGF receptor, with the mature erbB-3 protein for signal transduction. This suggests that within the erbB/EGFR family. the erbB-3 protein may function as a distinct growth factor receptor for an as yet unidentified ligand.